Method for improving antibody

ABSTRACT

The present invention provides for a method for improving properties of an antibody such as an expression level and stability. A method for obtaining an antibody with an improved expression level and/or stability by modifying human antibody or a humanized antibody, characterized by that at least any one of the amino acid residues at position 8, 12, 15 or 18 (according to Kabat numbering) in a light chain variable region (hereinafter referred to as “VL chain”) of a human antibody or a humanized antibody is substituted with a different amino acid other than proline or cysteine, and a human antibody or a humanized antibody or a human antibody fragment or a humanized antibody fragment with an improved expression level and/or stability which are obtained by said method.

TECHNICAL FIELD

The present invention relates to a technique for improving properties ofa human antibody such as an expression level and stability throughmolecular modification of the antibody molecule by amino acidsubstitution. The technique according to the present invention isexpected to be a means for tackling problems such as a lowered level ofproduction, or aggregation, denaturation or inactivation when handling,which have been a great burden to research and development with anantibody.

BACKGROUND ART

An antibody medicament, which is based on the mechanism of biologicalprotection in human, is highly expected since it is a moleculartargeting therapeutics which targets a specific functional molecule. Itshigh efficacy shown in the field of cancer and rheumatism, inter alia,has boosted the world-wide development rush in recent years and itsworld market scale is thought to keep magnifying for the time being.

Under such social background, marked progress in the technique has beendeveloped in the field of an antibody medicament and antibodyengineering. However, some of the obtained clones of antibody moleculeshave posed much difficulty in their handling due to a low level ofexpression and/or stability and its solution is not so easy a matter.The problems stated hereinabove have been a great burden to research anddevelopment with an antibody.

Another problem confronted in case of an antibody medicament is itscost. Since a comparatively large quantity of antibodies is required forthe therapy and also heavy investment in plant and equipment occurs,increase in not only a burden of patients but also in national expensesfor medical treatment will result. Accordingly, an important propositionfor the development of an antibody medicament is to improve productivityand to lower its cost.

In the living body, an antibody sequence is produced by a random geneticrecombination or mutagenesis occurring while maturation of B cells,among which one having an optimized antibody sequence selected andproliferated. The optimization chiefly depends upon an antigen-bindingcapacity. However, a number of factors including stability of domains,interaction between H chain and L chain, interaction between a variableregion and a constant region, a protease sensibility and secretionefficiency are thought to complicatedly attribute to the optimization.Thus, natural antibody sequences are not necessarily optimized withrespect of stability.

If an isolated antibody clone is expressed as a recombinant protein foranalysis, it is sometimes found to be fairly unstable. As a consequence,various problems arise, i.e. (1) an expression level is extremely low;(2) it is expressed not in a soluble form but as inclusion bodies tothereby necessitate refolding; (3) exposure to an acid whilepurification causes denaturation; or (4) precipitation and denaturationoccurs with standing at room temperature or even at 4° C. and thus thereactivity disappeared.

For resolving these problems, the first approach may be investigation ofoptimal conditions of operations or buffers for respective antibodyclones. However, the investigation would not only require much labor andcost but in some cases, by failure of dissolving the problems, mightdrive a research worker into giving-up of the subsequent analysis ordevelopment of an antibody clone which may have had a promisingreactivity.

The second approach may be a molecular modification such as amino acidsubstitution or conversion of a molecular form for aiming at theimprovement of the properties of an antibody. In general, modificationfor improving protein stability includes transplantation more conservedamino acids from other homologous sequences, and rational design viacomputer modeling for increasing hydrophilicity on the molecular surfaceor increasing hydrophobicity within the interior of hydrophobic core toenhance the strength of packing (Non-patent reference 1).

With respect to the improvement of stability or an expression level ofan antibody molecule via amino acid substitution, many of previousreports are concerned with characteristic amino acid modification forsequences of respective antibody clones but there are known only fewtechniques for modification that may be generalized. Such techniques formodification capable of generalization includes amino acid substitutionat one or two sites in VH to improve stability of a single chainantibody (scFv) as reported by Worn et. al. (Non-patent reference 2);amino acid substitution at one or plural sites in VH to improve anexpression level of Fv fragments and to inhibit aggregation reaction asreported by Knappik et al. (Non-patent reference 3); amino acidsubstitution at one or two sites in VL to improve stability of a VLdomain as reported by Steipe et al. (Non-patent reference 4); and aminoacid substitution at position 34 (according to Kabat numbering) in VL toimprove stability and an expression level of scFv as reported by Hugo etal. (Non-patent reference 5). However, the objective antibodies subjectto modification by all these reports are mouse antibodies.

For the use as an antibody medicine, a mouse antibody is recognized as aforeign substance and excluded due to its high antigenicity whenadministered to human. Thus, it would be difficult to use a mouseantibody as a medicament for therapy of diseases. For dissolving thisproblem, a mouse monoclonal antibody may be converted to a chimericantibody using a protein engineering technique. However, a mousechimeric antibody still contains sequences derived from mice amountingto 30% or more and thus its repetitive or prolonged administration willlead to production of an antibody that inhibits the activity of thechimeric antibody administered to thereby not only extremely lower theefficacy of the medicament but to induce severe adverse side effects.

Therefore, main approach for solving these problems is to construct ahumanized antibody wherein a complementarity determining regions (CDRs)from a mouse variable region are transplanted into a human variableregion or a human antibody having sequences completely derived fromhuman.

On the other hand, there is a report by Ewert et al. (Non-patentreference 6) in which amino acid substitution is done for a humanantibody at one or plural sites in VH to allow for the improvement ofstability and an expression level of scFv. However, the human antibodymodified by Ewert et al. is of VH6 family and a frequency in use of thegenes belonging to said family is as low as 1.4 to 2.4% (Non-patentreference 7), which renders their technique not being widely feasibleone.

As described above, since a specific antibody targeting adisease-related antigen is extremely useful in the clinical field suchas human diagnosis and therapy, establishment of the technique forpreparing an antibody possessing all the features of high asspecificity, low immunogenicity and high productivity has earnestly beendesired.

-   Non-patent reference 1: Vriend et al., J. Comput. Aided Mol. Des.,    7(4), p. 367-396 (1993)-   Non-patent reference 2: Worn et al., Biochemistry, 37, p.    13120-13127 (1998)-   Non-patent reference 3: Knappik et al., Protein Eng., 8(1), p. 81-89    (1995)-   Non-patent reference 4: Steipe et al., J. Mol. Biol., 240, p.    188-192 (1994)-   Non-patent reference 5: Hugo et al., Protein Eng., 16(5), p. 381-386    (2003)-   Non-patent reference 6: Ewert et al., Biochemistry, 42, p. 1517-1528    (2003)-   Non-patent reference 7: Brezinschek et al., J. Clin. Invest.,    99(10), p. 2488-2501 (1997)

DISCLOSURE OF THE INVENTION Technical Problem to be Solved by theInvention

A technical problem to be solved by the present invention is toestablish a general method for improving an antibody starting from ahuman antibody or a humanized antibody, which are known to exhibit lowimmunogenicity to human, so as to obtain an antibody with highproductivity and excellent stability while low immunogenicity andspecific binding capacity are maintained.

Means for Solving the Problems

Under the circumstances, the present inventors have earnestly continuedresearch activities and as a result succeeded in significantly improvingexpression and stability of an antibody by substituting an amino acid oramino acids in a light chain variable region (VL chain) of a humanantibody with a different amino acid sequence to thereby complete thepresent invention. More specifically, the present invention ischaracterized by that at least any one of the amino acid residues atposition 8, 12, 15 or 18 (according to Kabat numbering) in a light chainvariable region (hereinafter referred to as “VL chain”) of a humanantibody is substituted with a different amino acid other than prolineor cysteine.

Thus, the present invention encompasses the inventions (1) to (18) asdescribed hereinbelow.

(1) A method for obtaining an antibody with an improved expression leveland for stability by modifying a human antibody or a humanized antibody,characterized by that at least any one of the amino acid residues atposition 8, 12, 15 or 18 (according to Kabat numbering) in a light chainvariable region (hereinafter referred to as “VL chain”) of a humanantibody or a humanized antibody is substituted with a different aminoacid other than proline or cysteine.(2) The method according to (1) wherein at least any one of proline atposition 8, 12, 15 or 18 or an amino acid residue at position 15 in a VLchain is substituted with a different amino acid other than proline orcysteine.(3) The method according to (1) or (2) wherein the amino acid residue atthe respective position after substitution is selected from any one ofthe amino acids as described below:

position 8: Gly, Thr, Arg or Ser

position 12: Ser, His, Val, Gly, Leu, Arg, Phe, Met or Glu

position 15: Arg, Ser, Gly or Phe

position 18: Arg, Ser, Phe, Ala, Trp, Leu or Gln.

(4) The method according to any one of (1) to (3) wherein said VL chainbelongs to any one of human Vκ1 family, human Vκ2 family or human Vκ3family.

(5) The method according to any one of (1) to (4) wherein the amino acidresidue at the respective position after substitution in the VL chainbelonging to human Vκ1 family is selected from any one of the aminoacids as described below:

position 8: Gly, Thr, Arg or Ser

position 15: Arg or Ser.

(6) The method according to (5) wherein FR1 of said VL chain belongingto human Vκ1 family prior to substitution has the sequence from DPK9(GeneBank Accession No. X59315).

(7) The method according to (5) or (6) wherein FR1 of the VL chainbelonging to human Vκ1 family after substitution has the amino acidsequence selected from the amino acid sequences as depicted in SEQ IDNO: 2 to 7.

(8) The method according to any one of (1) to (4) wherein the amino acidresidue at the respective position after substitution in the VL chainbelonging to human Vκ2 family is selected from any one of the aminoacids as described below:

position 12: Ser, His, Val, Gly, Leu, Arg, Phe, Met or Glu

position 15: Arg

position 18: Arg, Ser, Phe, Ala, Trp, Leu or Gln.

(9) The method according to (8) wherein FR1 of said VL chain belongingto human Vκ2 family prior to substitution has the sequence from DPK18(GeneBank Accession No. X63403).

(10) The method according to (8) or (9) wherein FR1 of the VL chainbelonging to human Vκ2 family after substitution has the amino acidsequence selected from the amino acid sequences as depicted in SEQ IDNO: 9 to 25.

(11) The method according to any one of (1) to (4) wherein the aminoacid residue at the respective position after substitution in the VLchain belonging to human Vκ3 family selected from any one of the aminoacids as described below:

position 15: Arg, Ser, Gly or Phe.

(12) The method according to (11) wherein FR1 of said VL chain belongingto human Vκ3 family prior to substitution has the sequence from DPK22(GeneBank Accession No. X59315).

(13) The method according to (11) or (12) wherein FR1 of the VL chainbelonging to human Vκ3 family after substitution has the amino acidsequence selected from the amino acid sequences as depicted in SEQ IDNO: 27 to 30.

(14) The method according to any one of (1) to (13) wherein saidantibody is an intact antibody, or an antibody fragment such as Fab,Fab′, F(ab′)₂, scAb, scFv, diabody [a recombinant dimer antibodyconsisting homologous or heterologous heavy chain variable region (VHchain) and VL chain connected by a short linker peptide] or scFv-Fc; ora fused antibody or a fused antibody fragment with other proteins; or anantibody or an antibody fragment labeled with a low molecular weightcompound; or an antibody or an antibody fragment modified with a highmolecular weight compound.(15) The method according to any one of (1) to (14) wherein the aminoacid substitution is done by a genetic recombination technique.(16) A human antibody or a humanized antibody or a human antibodyfragment or a humanized antibody fragment with an improved expressionlevel and/or stability which are obtained by the method as set forth inany one of (1) to (14).(17) A method for preparing an antibody with an improved expressionlevel and/or stability in which at least any one of the amino acidresidues at position 8, 12, 15 or 18 (according to Kabat numbering) in alight chain variable region (hereinafter referred to as “VL chain”) of ahuman antibody or a humanized antibody or a human antibody fragmenthumanized antibody fragment is substituted with a different amino acidother than proline or cysteine, which comprises:

preparing a gene coding for an amino acid sequence of said humanantibody or said humanized antibody or said human antibody fragment orsaid humanized antibody fragment, each comprising the amino acidsequence of the VL chain after substitution;

transforming a host of eukaryotic or prokaryotic organisms with saidgene;

expressing said human antibody or said humanized antibody or said humanantibody fragment or said humanized antibody fragment from said host;and

recovering said human antibody or said humanized antibody or said humanantibody fragment or said humanized antibody fragment.

(18) A human antibody or a humanized antibody or a human antibodyfragment or a humanized antibody fragment with an improved expressionlevel and/or stability which are prepared by the method as set forth in(17).

More Efficacious Effects than Prior Art

According to the present invention, as a consequence of molecularmodification of a human antibody molecule via amino acid substitution,it becomes possible to extremely improve the properties of an antibodysuch as its expression level and stability. The technique according tothe present invention is usable as a means for tackling the problemssuch as a lowered level of production, or aggregation, denaturation orinactivation when handling, which have been a great burden to researchand development with an antibody.

Furthermore, the method for improving an antibody according to thepresent invention may widely be used for the development of an antibodymedicine as a diagnostic or therapeutic agent since it aims atmodification of an amino acid residue in a framework region startingfrom a human antibody or a humanized antibody. Also, since only a singleamino acid residue is modified, a concern about antigenic induction whenadministered to human may be minimized.

Viewing that the improvement of heat stability through molecularmodification is reported to increase targeting of tumors in vivo(Willuda et al., Cancer Res., 59, p. 5758-5767 (1999)), it is expectedthat the improvement of stability leads to the increase in efficacy. Assuch, the method for improving an antibody according to the presentinvention is expected to much contribute to the development of anantibody therapy. Furthermore, since an antibody may be applied to notonly the medical field but also other various fields, the presentinvention is expected to be widely applicable to various fields such asrelated researches or progress of industry.

Accordingly, a human antibody or a humanized antibody or human antibodyfragment or a humanized antibody fragment obtained by the method of thepresent invention excels in an expression level and/or stability, maydecrease damage due to the loss of the activity or the formation ofaggregation during a purification step, and may provide utility inantibody production or purification step. Besides, as a matter ofcourse, since the method of the present invention may provide a humanantibody molecule or a humanized antibody molecule with excellentstability, it would be able to provide many choices in design ofpharmaceutical preparations and thus great advantage for theirproduction.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a structure of the plasmid used for expression of Fab.

FIG. 2 is a graph showing comparison between a wild-type SEB3-2-7 andits modified antibody at position 8 in L chain for their expressionlevel of a functional Fab protein in periplasm fractions in ELISA.

FIG. 3 is a graph showing comparison between a wild-type SEB3-2-7 andits modified antibodies at position 8 in L chain for their heatstability when treated at 42° C. in ELISA.

FIG. 4 is a graph showing comparison between a wild-type SEB3-2-7 andits modified antibodies at position 8 in L chain for their toleranceagainst an acid when treated at pH 4.5 in ELISA.

FIG. 5 is a graph showing comparison between a wild-type RNOK203 and itsmodified antibody at position 12 in L chain for their expression levelof a functional Fab protein in culture supernatant in ELISA.

FIG. 6 is a graph showing comparison between a wild-type RNOK203 and itsmodified antibodies a position 12 in L chain for their heat stabilitywhen treated at 40° C. in ELISA.

FIG. 7 is a graph showing comparison between a wild-type RNOK203 and itsmodified antibody at position 12 in L chain for their tolerance againstan acid when treated at pH 4.0 in ELISA.

FIG. 8 is a graph showing comparison between a wild-type RNOK203 and itsmodified antibodies at position 12 in L chain for their toleranceagainst freeze-thawing in ELISA.

FIG. 9 is a graph showing comparison between a wild-type CTLA4-3-1 andits modified antibody at position 15 in L chain for their expressionlevel of a functional Fab protein in periplasm fractions in ELISA.

FIG. 10 shows structure of the expression plasmids for wild-typeCTLA4-3-1 Fab, in which a synthetic Oligo DNA coding for E tag isinserted at the C-terminal of Fd chain, and its P15R modified antibody.Panels (a) and (b) show wild-type CTLA4-3-1 Fab and its P15R modifiedantibody, respectively.

FIG. 11 is a graph showing comparison between a wild-type CTLA4-3-1Fab-E and its P15R modified antibody for their expression level of Fdchain and L chain in culture supernatant fractions and periplasmfractions in Western blotting. Panel (a) shows the results for Fd chainwhich was detected with HRP-labeled anti-E tag Ab. Panel (b) shows theresults for L chain which was detected with HRP-labeled anti-c-myc tagAb. Lane 1: marker; Lane 2: wild-type CTLA4-3-1 Fab-E in culturesupernatant fractions; Lane 3: P15R modified CTLA4-3-1 Fab-E in culturesupernatant fractions; Lane 4: wild-type CTLA4-3-1 Fab-E in periplasmfractions; and Lane 5: P15R modified CTLA4-3-1 Fab-E in periplasmfractions.

FIG. 12 is a graph showing comparison between a wild-type CTLA4-3-1Fab-E and its P15R modified antibody for their expression level of Fdchain/L chain complex in culture supernatant fractions and periplasmfractions in sandwich ELISA. Panels (a) and (b) show the results forculture supernatant fractions and periplasm fractions, respectively.

FIG. 13 is a graph showing comparison between a wild-type CTLA4-3-1 andits modified antibodies at position 15 in L chain for their heatstability when treated at 28° C. in ELISA.

FIG. 14 is a graph showing comparison between a wild-type CTLA4-3-1 andits modified antibodies at position 15 in L chain for their toleranceagainst an acid in ELISA. Panels (a) and (b) show the results whentreated at pH 4.0 and at pH 3.5, respectively.

FIG. 15 is a graph showing comparison between a wild-type RNOK203 andits modified antibody at position 15 in L chain for their expressionlevel of a functional Fab protein in culture supernatant fractions inELISA.

FIG. 16 is a graph showing comparison between a wild-type RNOK203 andits modified antibody at position 15 in L chain for their toleranceagainst an acid when treated at pH 5.0 in ELISA.

FIG. 17 is a graph showing comparison between a wild-type SEB3-2-7 andits modified antibodies at position 15 in L chain for their heatstability when treated at 42° C. in ELISA.

FIG. 18 is a graph showing comparison between a wild-type SEB3-2-7 andits modified antibodies at position 15 in L chain for their toleranceagainst an acid in ELISA. Panels (a) and (b) show the results whentreated at pH 4.5 and at pH 4.0, respectively.

FIG. 19 is a graph showing comparison between a wild-type RNOK203 andits modified antibodies at position 18 in L chain for their heatstability when treated at 45° C. in ELISA.

FIG. 20 is a graph showing comparison between a wild-type RNOK203 andits modified antibodies at position 18 in L chain for their toleranceagainst an acid when treated at pH 4.0 in ELISA.

FIG. 21 is a graph showing comparison between a wild-type RNOK203 andits modified antibodies at position 18 in L chain for their toleranceagainst freeze-thawing in ELISA.

BEST MODE FOR CARRYING OUT THE INVENTION

VL chain of a human antibody to be modified includes λ chain and κchain, among which κ chain may preferably be used by reasons that 97% ofmouse L chain is κ chain (“Menekigaku Citen” (Dictionary of Immunology),2nd Ed., 2001, TOKYO KAGAKU DOJIN CO., LTD.) and thus human Vκ is usedas a template sequence in most cases of humanization technique and thatκ chain is also major, i.e. 67%, human L chain (Knappik et al., J. Mol.Biol., 296, p. 57-86 (2000)).

In addition, it is known that human Vκ includes families of Vκ1 to Vκ6.A frequency in use of Vκ1, Vκ2 and Vκ3 in total amounts to 92% to covermost of human Vκ (Foster et al., J. Clin. Invest. 99(7), p. 1614-1627(1997)). Thus, any of these three families, Vκ1, Vκ2 and Vκ3, maypreferably be used.

As a strategy for antibody modification, amino acid substitution wasinvestigated targeting proline (Pro; P), the amino acid which is in aframework (FR) 1 region important for maintenance of steric structure ofVL chain of a humanized antibody and is known to be rate-determining forfolding and to destroy α helix and β sheet structures.

Specifically, any one of the amino acid residues at position 8, 12, 15or 18 in VL chain of a human antibody or a humanized antibody wastargeted for modification. As used herein, the identification of theposition of amino acid residues in VL chain of an antibody was done inaccordance with the numbering scheme by Kabat et al. (Kabat, E. A. etal., Sequences of Proteins of Immunological Interest, 5th Ed., NIHPublication No. 91-3242, 1991).

For modification, most of the previous reports were concerned withapproach rational design as described above. As for the technique bytransplantation of highly conserved amino acid residues, an antibodysequence as selected in nature is the consequence of choice viacomplicated attribution of a number of factors such as stability ofdomains, interaction between H chain and L chain, interaction between avariable region and a constant region, a protease sensibility andsecretion efficiency out of cells. Thus, an antibody sequence in natureis not necessarily optimized with respect to stability and hence thistechnique has limitation. Also, approach using computer modeling notguarantee actual stability in spite of much labor and thus is not anefficient approach at present.

The present inventors thus investigated by evolutionary engineeringtechnique, i.e. substituted the amino acid residues targeted formodification with any amino acid at random and selected modifiedantibodies with desired properties from the group obtained.

As a result, as described in Examples hereinbelow, it was verified thatthe improvement of an expression level and stability could be observedwhen any one of the following amino acid substitutions was induced inFR1 (1st to 23rd) of VL chain:

position 8: Gly, Thr, Arg or Ser

position 12: Ser, His, Val, Gly, Leu, Arg, Phe, Met or Glu

position 15: Arg, Ser, Gly or Phe

position 18: Arg, Ser, Phe, Ala, Trp, Leu or Gln.

Original sequences (wild) of FR1 of VL chain prior to substitutiontypically include the following amino acid sequences:

Vκ1 (Anti-SEB Ab SEB3-2-7): (SEQ ID NO: 1) DIVMTQSPSSLSASVGDTVTITCVκ2 (Anti-FasL Ab RNOK203): (SEQ ID NO: 8) DVVMTQTPLSLPVTLGQPASISCVκ3 (Anti-CTLA-4 Ab CTLA4-3-1): (SEQ ID NO: 26) EIVLTQSPGTLSLSPGERATLSC

More specifically, modification was performed as described below inaccordance with the present invention and the obtained antibodies wereassessed for their expression level and stability (i.e. toleranceagainst heat, tolerance against an acid, tolerance againstfreeze-thawing, etc.) to prove remarkable improvement in all theobtained antibodies. As used hereinbelow, the symbol “-” means the aminoacid residue that is identical to that of a wild-type antibody.

(1) Vκ1 (SEQ ID NO: 1) Wild: DIVMTQSPSSLSASVGDTVTITC (SEQ ID NO: 2) P8G:-------G--------------- (SEQ ID NO: 3) P8T: -------T---------------(SEQ ID NO: 4) P8R: -------R--------------- (SEQ ID NO: 5) P8S:-------S--------------- (SEQ ID NO: 6) V15R: --------------R--------(SEQ ID NO: 7) V15S: --------------S-------- (2) Vκ2 (SEQ ID NO: 8)Wild: DVVMTQTPLSLPVTLGQPASISC (SEQ ID NO: 9) P12S:-----------S----------- (SEQ ID NO: 10) P12H: -----------H-----------(SEQ ID NO: 11) 912V: -----------V----------- (SEQ ID NO: 12) P12G:-----------G----------- (SEQ ID NO: 13) P12L: -----------L-----------(SEQ ID NO: 14) P12R: -----------R----------- (SEQ ID NO: 15) P12F:-----------F----------- (SEQ ID NO: 16) P12M: -----------M-----------(SEQ ID NO: 17) P12E: -----------E----------- (SEQ ID NO: 18) P15P:--------------R-------- (SEQ ID NO: 19) P18R: -----------------R-----(SEQ ID NO: 20) P18S: -----------------S----- (SEQ ID NO: 21) P18F:-----------------F----- (SEQ ID NO: 22) P18A: -----------------A-----(SEQ ID NO: 23) P18W: -----------------W----- (SEQ ID NO: 24) P18L:-----------------L----- (SEQ ID NO: 25) P18Q: -----------------Q-----(3) Vκ3 (SEQ ID NO: 26) Wild: EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 27)P15R: --------------R-------- (SEQ ID NO: 28) P15S:--------------S-------- (SEQ ID NO: 29) P15G: --------------G--------(SEQ ID NO: 30) P15F: --------------F--------

Any of the modifications as specified above have neither been reportednor practiced in prior patents for antibodies derived from animals, letalone for antibodies from human, and thus are firstly investigated inthe present application.

In particular, the modified antibody (P15R) with substitution to Arg (R)at position 15 in L chain exhibited drastic effect, namely about100-fold increase in an expression level of a functional molecule. Somuch effect with a single amino acid substitution in Fab has never beenreported so far. Furthermore, it was demonstrated that the technique formodification for improving an expression level or stability bysubstituting the 15th amino acid residue in L chain to Arg or Ser may beapplicable to at least Vκ1, Vκ2 and Vκ3 families and thus be of wideuse.

For the amino acid substitution in accordance with the presentinvention, modified antibody libraries wherein an arbitrary amino acidwas introduced at random at position 8, 12, 15 or 18 in VL chain wereconstructed using a model antibody. For a model antibody, a humananti-SEB antibody (a clone called “SEB3-2-7”), a human anti-CTLA-4antibody (a clone called “CTLA4-3-1”) and a human anti-human FasLantibody RNOK were used for each of Vκ1, Vκ2 and Vκ3 families,respectively. Using Oligo DNA primers in which a codon of a targetedamino acid in a VL chain gene of the respective antibodies was replacedwith a random codon NNK wherein N is A or C or G or T, and K is G or T,a gene of a mutated VL chain was amplified by PCR using a gene ofwild-type VL chain as a template. The thus amplified gene of a mutatedVL chain was replaced for the VL region in a wild-type Fab expressionplasmid to construct a modified Fab expression plasmid. JM83 wastransformed with the obtained plasmid, many clones were obtained andisolated, and expression was induced on 96-well microtiter plate. Theexpression of Fab periplasm fractions was assessed by Dot Blot andclones where expression was verified were analyzed for their properties.Fab is an antibody molecule that may be expressed soluble in E. coli andconvenient for the analysis of properties of a variable region.

By comparison of an expression level of modified Fabs, clones with ahigher expression level than that of wild-type Fab were analyzed fortheir DNA nucleotide sequence to prove that they each contain a singleamino acid substitution. The clones were further assessed for theirexpression level, heat stability, tolerance against an acid andtolerance against freeze-thawing.

An expression level may be assessed and compared between the clones e.g.by inducing expression in shaker-flask culture, and assessing theabsorbance of recovered culture supernatant or periplasm fractions byusing ELISA as an amount of a functional Fab protein.

Heat stability may be assessed e.g. by diluting and treating with awater-bath at a specified temperature for 2 hours the culturesupernatant fractions or the periplasm fractions in which Fab wasexpressed, restoring the samples at room temperature, performing ELISA,and assessing and comparing between the clones a ratio of the obtainedabsorbance to that of untreated samples as a residual activity.

Tolerance against an acid may be assessed e.g. by diluting and adjustingto a specified pH for 2 hours the culture supernatant fractions or theperiplasm fractions in which Fab was expressed, neutralizing thesamples, performing ELISA, and assessing and comparing between theclones ratio of the obtained absorbance to that of untreated samples asa residual activity.

Tolerance against freeze-thawing may be assessed e.g. repeatingfreeze-thawing for specified times for the culture supernatant fractionsor the periplasm fractions in which Fab was expressed, performing ELISA,and assessing and comparing between the clones a ratio of the absorbanceof the samples subjected to plural freeze-thawing procedures to that ofsamples subjected to one freeze-thawing procedure as a residualactivity.

As a consequence of the assessment of modification, the presentinventors have found that the modification as detailed above couldimprove the properties of an antibody.

In accordance with the present invention, a prepared modified antibodymay be utilized not only as an intact antibody but also in the form ofan antibody fragment such as Fab, Fab′, F(ab′)₂, scAb, scFv, diabody orscFv-Fc. Such an antibody or an antibody fragment may also be anantibody fused with other proteins or peptides. The expressed antibodymay also be labeled with various chemicals or radioactive elements ormodified with a synthetic high molecular weight compound suchpolyethylene glycol.

An antibody or an antibody fragment modified in accordance with thepresent invention may be prepared, using a genetic recombinationtechnique and based on sequence information of genes coding for saidantibody or antibody fragment, by introducing said genes into a suitablehost, e.g. bacteria, Bacillus subtilis, yeast, animal cells, andexpressing said antibody or antibody fragment.

As used herein, “a humanized antibody” refers a human antibody whereinonly its complementarity determining regions (CDRs) in a variable regionare derived from that of a non-human animal antibody but other variableregion than CDR, i.e. a framework region on (FR), as well as a constantregion are derived from a human antibody.

The present invention is explained in more detail by means of thefollowing Examples but should not be construed to be limited thereto.

Example 1: E. Coli Strain and DNA

(1) E. coli Strain

JM83 strain (Gene, 33, p, 103-119 (1985)) was used.

(2) Plasmid

Plasmid pTrc99A-Fab-myc codes for each of Fab clones under the controlof trc promoter. For allowing for more efficient expression and foldingin E. coli, the Cys residues at the C-terminal of CH1 and Cκ regions aresubstituted with Ser to thereby render the molecule lacking disulfidebond between the chains. For easier detection, c-myc tag is added at theC-termanal or L chain. For secretion into periplasm and culturesupernatant, pelB sequence signals (Lei et al., J. Bacteriol., 169, p.4379-4383 (1987)) are inserted upstream both the coding regions of bothchains (FIG. 1).

(3) Oligo DNA

Oligo DNAs as used herein were those from SIGMA GENOSYS.

Example 2: Antibody Clones

(1) Anti-SEB Antibody SEB3-2-7

This antibody is a human antibody capable of specifically recognizingSEB which was isolated by panning with a recombinant SEB protein fromscFv display phage library constructed using as a starting materialperipheral blood lymphocytes from 20 healthy volunteers. Its VH has asequence derived from a segment DP-75 belonging to VH1 family and its VLhas a sequence derived from a segment DPK9 (GenBank Accession No.X59315) belonging to Vκ1 family. The amino acid sequence of FR1 of VLdomain is shown in SEQ ID NO: 1.

(2) Anti-FasL Antibody RNOK203

This antibody is humanized antibody which specifically recognizes Fasligand and has a neutralizing activity as reported by Nakashima et. al.,J. Immunol., 167, p. 3266-3275 (2001). Its VL has a sequence derivedfrom a segment DPK18 (GenBank Accession No. X63403) belonging to Vκ2family. The amino acid sequence of FR1 of VL domain is shown in SEQ IDNO: 8.

(3) Anti-CTLA-4 Antibody CTLA4-3-1

This antibody is a human antibody capable of specifically recognizingCTLA-4 which was isolated by panning with a recombinant CTLA-4 proteinfrom scFv display phage library constructed using as starting materialperipheral blood lymphocytes from 20 healthy volunteers. Its VH has asequence derived from a segment DP-25 belonging to VH1 family and its VLhas a sequence derived from a segment DPK22 (GenBank Accession No.X59315) belonging to Vκ3 family. The amino acid sequence of FR1 of VLdomain is shown in SEQ ID NO: 26.

Example 3: Construction of Wild-Type Fab Expression Plasmid

In contrast to the prior art or the prior reports as described above,most of which used VL domain alone or scFv, modification of an antibodywas investigated using Fab in accordance with the present invention. Anantibody fragment is advantageous in that it may be expressed inbacteria allowing for quick investigation as compared to IgG which willneed expression in an animal cell to thereby necessitate much time andlabor for analysis. For a domain alone or scFv, it is known that achange of properties such as lowered antigen affinity, possibly due tostructural change, sometimes occurs when sequences of VH and VL arerecombined to form IgG molecule but such a change is not likely to occurwhen Fab is converted to IgG molecule. Accordingly, it is highlypossible that effect of modification observed for Fab molecule may beretained even after the Fab molecule is converted into its correspondingIgG molecule. Viewing that most of the currently available antibodymedicines is in the form of IgG molecule, the technique for improving anantibody according to the present invention is expected to muchcontribute to the field of an antibody medicine.

PCR was performed with Pyrobest DNA Polymerase (TAKARA) foramplification or VH gene and VL gene using scFv plasmid (vector;pCANTAB5E) for SEB3-2-7 and CTLA4-3-1 and IgG expression vector (vector;pCAG: Gene 108, p. 193-200, 1991) for RNOK203 as starting material. TheVH gene region digested with SfiI and the VL gene region digested withXhoI and NotI were inserted into pTrc99A-Fab-myc vector in order.

Example 4: Transformation

Transformation was performed by electroporation using Gene Pulser(BIO-RAD). JM83 strain was made competent, transformed, applied to LBagar medium containing 50 μg/mL ampicillin, and cultured at 30° C.overnight. The obtained clones were isolated and cultured. The plasmidswere prepared by the conventional manner and the DNA sequences wereanalyzed.

Example 5: DNA Sequencing

A DNA sequence of the modified VL gene was determined using CEQ DTCSQuick Start Kit (BECKMAN COULTER). For the expression strain asconstructed, the DNA sequencing proved that it contained the sequence asdesigned.

Example 6: Construction of Modified Fab Expression Plasmids

Using a wild-type Fab expression plasmid as a template, PCR wasperformed as described above for amplification of VL gene using OligoDNAs in which codon at the site for mutation was NNK wherein N is A or Cor G or T, and K is G or T. The amplified VL gene was replaced for theVL region of a wild-type Fab expression plasmid. JM83 was transformedwith the resulting plasmids and expression was induced for the obtainedclones an 96-well microtiter plate.

Example 7: Induction of Fab Expression by Plating Culture

The clones obtained above and a wild-type Fab-expressing strain as acontrol were inoculated to 2×YT culture containing 50 μg/mL ampicillinon 96-well plate (COSTAR) and cultured at 30° C. for 5 to 6 hours. IPTGwas added to the plate at a final concentration of 1 mM and culture wascontinued overnight to induce Fab expression. After completion ofculture, the cells were centrifuged and recovered, suspended in PBScontaining 1 mM EDTA and left to stand on ice for 30 minutes. The cellswere then centrifuged at 2,000 rpm for 15 minutes and the supernatantwas recovered to obtain a periplasm fraction.

Example 8: Dot Blot

Expression level was analyzed by Dot Blot. The periplasm fractionobtained above was spotted on 0.45 μm nitrocellulose filter (Millipore)and, following blocking with PBS-0.05% Tween20 containing 2% skim milk,Fab expression was detected with peroxidase (HRP)-labeled anti-Fab Ab(CAPPEL). For those clones which were thought to exhibit a higherexpression level, the DNA sequences were analyzed as described above andthe clones which could prove to undergo single amino acid substitutionwere assessed as described hereinbelow.

Example 9: Induction of Fab Expression by Shaking Culture

The suspension of E. coli cells was applied to LB agar containing 50μg/mL ampicillin and cultured at 30° C. overnight. The obtained singlecolony was then inoculated to 2×YT culture containing 50 μg/mLampicillin and cultured at 30° C. until O. D. at 600 nm becomes 0.5 to1.0. IPTG was added at a final concentration of 1 mM and culture wascontinued overnight to induce Fab expression. After completion ofculture, the cells were centrifuged and the supernatant was recovered toobtain a supernatant fraction. The precipitated cells were suspended inPBS containing 1 mM EDTA and left to stand on ice for 30 minutes. Thecells were then centrifuged at 8,900×g for 15 minutes and thesupernatant was recovered as a per fraction.

Example 10: Assessment of Reactivity with SEB by ELISA

The expressed and purified recombinant SEB at 200 ng/100 μL/well wasimmobilized on an ELISA plate (Nunc) at 4′C. overnight and, afterwashing, blocked with 1% BSA-PBS at 4° C. overnight. The periplasmfractions or the culture supernatant fractions of wild-type SEB3-2-7/itsmodified antibody were diluted to 100 μL/well with 1% BSA-PBS andreacted at 37° C. for 1 hour. Detection was performed with a combinationof biotin-labeled anti-Kappa Ab (Southern Biotechnology) and peroxidase(HRP)-labeled streptavidin (Vector Lab.). Absorbance at 650 nm/450 nmwas measured with Microplate Reader Vmax (Molecular Devices).

Example 11: Assessment of Expression Level of Modified Antibodies atPosition 8 in L Chain with SEB3-2-7

The periplasm fractions of a wild-type SEB3-2-7/its modified antibodieswere diluted step-wise and compared for their expression level of afunctional Fab protein in ELISA. As a result, it was found that P8Gmodified antibody exhibited an increased expression level (FIG. 2).

Example 12: Assessment of Heat Stability of Modified Antibodies atPosition 8 in L Chain with SEB3-2-7

The culture supernatant fractions of wild-type SEB3-2-7/its modifiedantibodies were diluted to 50 μL/tube with 1% BSA-PBS and treated in awater-bath at 42° C. for 2 hours. The samples were then restored at roomtemperature and subjected to ELISA where a ratio of the obtainedabsorbance to that of untreated samples was shown as a residual activityin a graph. As a result, it was found that P8T, P8G, P8R and P8Smodified SEB3-2-7 antibodies exhibited increased heat stability (FIG.3).

Example 13: Assessment of Tolerance Against Acid of Modified Antibodiesat Position 8 in L Chain with SEB3-2-7

The Culture supernatant fractions of wild-type SEB3-2-7/its modifiedantibodies were diluted to 50 μL/tube with 1% BSA-PBS and adjusted to pH4.5 with 1N HCl and a pH meter (HORIBA) and treated at 25° C. for 2hours. The samples were then adjusted to pH 7 with 1M Tris-HCl (pH 9.5)and subjected to ELISA where a ratio of the obtained absorbance to thatof untreated samples was shown as a residual activity in a graph. As aresult, it was found that P8T, P8G, P8R and P8S modified SEB3-2-7antibodies exhibited increased tolerance against an acid (FIG. 4).

Example 14: Assessment of Reactivity with FasL by ELISA

For FasL, a commercially available recombinant Fas Ligand (R&D systems)with His-tag at 50 ng/100 μL/well was immobilized on an NI chelate plate(Nunc) at room temperature for 2 hours. The culture supernatantfractions of wild-type RNOK203/its modified antibody were diluted to 100μL/well with Block Ace (Dainippon Pharma Co., Ltd.) and reacted at 37°C. for 1 hour. Detection was performed with a combination ofbiotin-labeled anti-Kappa Ab (Southern Botechnology) and peroxidase(HRP)-labeled streptavidin (Vector Lab.). Absorbance at 650 nm/450 nmwas measured with Microplate Reader Vmax (Molecular Devices).

Example 15: Assessment of Expression Level of Modified Antibodies atPosition 12 in L Chain with RNOK203

The culture supernatant fractions of a wild-type RNOK203/its modifiedantibodies were diluted step-wise and compared for their expressionlevel of a functional Fab protein in ELISA. As a result, it was foundthat P12S modified antibody exhibited an increased expression level(FIG. 5).

Example 16: Assessment of Heat Stability of Modified Antibodies atPosition 12 in L Chain with RNOK203

The culture supernatant fractions of wild-type RNOK203/its modifiedantibodies were diluted to 50 μL/tube with Block Ace and treated in awater-bath at 40° C. for 2 hours. The samples were then restored at roomtemperature and subjected to ELISA where a ratio of the obtainedabsorbance to that of untreated samples was shown as a residual activityin a graph for comparison between the modified antibodies. As a result,it was found that P12S, P12H, P12V, P12G, P12L, P12R, P12F, P12M andP12E modified RNOK203 antibodies exhibited increased heat stability(FIG. 6). Typical results are shown in a graph (FIG. 6) whereas theresults of the remaining modified antibodies summarized in Table 1 for aratio of the respective residual activity to that of the wild-typeantibody.

TABLE 1 Comparison with wild- Modified antibodies type (%) P12S 140 P12H150 P12V 270 P12G 230 P12L 250 P12R 190 P12F 170 P12M 140 P12E 210

Example 17: Assessment of Tolerance Against Acid of Modified Antibodiesat Position 12 in L Chain with RNOK203

The culture supernatant fractions of wild-type RNOK203/its modifiedantibodies were diluted to 50 μL/tube with Block Ace and adjusted to pH4.0 with 1N HCl and a pH meter (HORIBA) and treated at 25° C. for 2hours. The samples were then adjusted to pH 7 with 1M Tris-HCl (pH 9.5)and subjected to ELISA where a ratio of the obtained absorbance to thatuntreated samples was shown as a residual activity in a graph. As aresult, it was found that P12V modified RNOK203 antibody exhibitedincreased tolerance against an acid (FIG. 7).

Example 18: Assessment of Tolerance Against Freeze-Thawing of ModifiedAntibodies at Position 12 in L Chain with RNOK203

The culture supernatant fractions of wild-type RNOK705/its modifiedantibodies were subjected to freeze-thawing repeated for either sixtimes or only once and then to ELISA where a ratio of the absorbanceobtained from the samples subjected to six freeze-thawing procedures tothat of samples subjected to only one freeze-thawing procedure as aresidual activity. As a result, it was found that P12S, P12V, P12G,P12L, P12R and P12F modified RNOK203 antibodies exhibited increasedtolerance against freeze-thawing (FIG. 8).

Example 19: Assessment of Reactivity with CTLA-4 by ELISA

For CTLA-4, a commercially available recombinant CTLA-4 (R&D systems) at100 ng/100 μL/well was immobilized on an ELISA plate (Nunc) at roomtemperature for 1 hour. After washing, the plate was blocked with BlockAce at room temperature for 1 hour. The periplasm fractions of wild-typeCTLA4-3-1/its modified antibody were diluted to 100 μL/well with BlockAce and reacted at 37° C. for 1 hour. Detection was performed with acombination of biotin-labeled anti-Kappa Ab (Southern Biotechnology) andperoxidase (HRP)-labeled streptavidin (Vector Lab.). Absorbance at 650nm/450 nm was measured with Microplate Reader Vmax (Molecular Devices).

Example 20: Assessment of Expression Level of Modified Antibodies atPosition 15 in L Chain with CTLA-4

The periplasm fractions of a wild-type CTLA4-3-1/its modified antibodieswere diluted step-wise and compared for their expression level of afunctional Fab protein in ELISA. As a result, it was found that P15R,P15S and P15G modified antibodies exhibited an increased expressionlevel FIG. 9). In particular, the P15R modified antibody exhibited adrastic effect of as high as about 100-fold increase in an expressionlevel. So much increase in an expression level of a functional Fabprotein with merely a single amino acid substitution has never beenreported so far.

Example 21: Construction of Wild-Type CTLA4-3-1 Fab-E/its P15R ModifiedAntibodies Expression Plasmid

In order to further verify the increased expression level of the P15Rmodified antibody as observed in Example 20, the Fab expression plasmidwas modified. Expression plasmids for wild-type CTLA4-3-1 Fab/its P15Rmodified Fab were constructed (FIG. 10) wherein a synthetic Oligo DNAcoding for E tag was inserted at the C-terminal of Fd chain (i.e. aregion of H chain including VH to CH1). Hereinafter, Fab containing Fdchain with E tag is referred to as “Fab-E”. JM83 strain as competentcells was transformed with the resulting plasmids and DNA sequencingproved that they contained the sequences as designed. Thus, it has nowbecome possible to detect Fd chain and L chain both constituting Fabeither by the use of E tag for Fd chain or c-myc tag for L chain.

The thus constructed wild-type CTLA4-3-1 Fab-E/its P15R modified Fab-Ewere subjected to induction of expression by shaking culture as inExample 9 and culture supernatant fractions and periplasm fractions wererecovered.

Example 22: Assessment of Expression Level of Wild-Type CTLA4-3-1Fab-E/its P15R Modified Fab-E by Western Blotting

The culture supernatant fractions and the periplasm fractions obtainedin Example 21 were analyzed by Western blotting to detect Fd chain and Lchain. Detection of Fd chain and L chain was performed with HRP-labeledanti-E tag Ab (Amersham Biosciences) and ERP-labeled anti-c-myc tag Ab(Roche), respectively. As a result, neither Fd chain nor L chain couldbe detected for the wild-type Fab-E whereas bands could be detected forthe P15R modified Fab-E at about 27 kDa and about 26 EPa for Fd chainand L chain, respectively, which virtually corresponded to the molecularweights as designed (FIG. 11). These results confirmed that the P15Rmodification increased expression level of both Fd chain and L chain.

Example 23: Assessment of Expression Level of Wild-Type CTLA4-3-1Fab-E/its P15R Modified Fab-E by Sandwich ELISA

In order to investigate whether Fd chain and L chain are assembledtogether to form a correct molecular form of Fab, the following sandwichELISA system was utilized. Anti-c-myc tag Ab 9E10 at 200 ng/100 μL/wellwas immobilized on an ELISA plate (Nunc) at room temperature for 1 hour.After washing, the plate was blocked with 1% BSA-PBS at room temperaturefor 1 hour. Each of the fractions were diluted to 100 μL/well with 1%BSA-PBS and reacted at 37° C. for 1 hour. Detection was performed withHRP-labeled anti-E tag Ab. Absorbance at 650 nm/450 nm was measured withMicroplate Reader Vmax (Molecular Devices).

As a result, it was found that, while no detection was observed forwild-type CTLA4-3-1 Fab-E, a concentration-dependent reaction wasobserved for P15R modified Fab-E with several ten times higher reactionfor the periplasm fractions thereof (FIG. 12). These results confirmedthat the P15R modification also highly increased an expression level ofa complex of Fd chain and L chain.

Example 24: Assessment of Heat Stability of Modified Antibodies atPosition 15 in L Chain with CTLA4-3-1

The periplasm fractions of wild-type CTLA4-3-1/its modified antibodieswere diluted to 50 μL/tube with Block Ace and treated in a water-bath at28° C. for 2 hours. The samples were then restored at room temperatureand subjected to ELISA where a ratio of the obtained absorbance to thatof untreated samples was shown as a residual activity in a graph. As aresult, it was found that P15R, P15S, P15G and P15F modified CTLA4-3-1antibodies exhibited increased heat stability (FIG. 13).

Example 25: Assessment of Tolerance Against Acid of Modified Antibodiesat Position 15 in L Chain with CTLA4-3-1

The periplasm fractions of wild-type CTLA4-3-1/its modified antibodieswere diluted to 50 μL/tube with Block Ace and adjusted to pH 4.0 or pH3.5 with 1N HCl and a pH meter (HORIBA) and treated on ice for 2 hours.The samples were then adjusted to pH 7 with 1M Tris-HCl (pH 9.5) andsubjected to ELISA where a ratio of the obtained absorbance to that ofuntreated samples was shown as a residual activity an a graph. As aresult, it was found that P15R, P15S, P15G and P15F modified CTLA4-3-1antibodies exhibited increased tolerance against an acid (FIG. 14).

Example 26: Construction of Modified Antibodies at Position 15 in LChain of RNOK203

It was investigated whether the same effects observed in Examples 20 to25 for the modification to Arg or Ser at position 15 in L chain couldalso be detected for RNOK203 wherein the 15th amino acid residue is Leu.

Using a wild-type Fab expression plasmid as template, PCR was performedas described above for amplification of VL gene using Oligo DNAs inwhich codon at position 15 in L chain was CGT(Arc) and TCT(Ser). Theamplified VL gene was replaced for the VL region of wild-type Fabexpression plasmid. JM83 was transformed with the resulting plasmid andthe DNA sequence was analyzed for the obtained clones to prove that theycontained the sequences as designed.

Example 27: Assessment of Expression Level of Modified Antibodies atPosition 15 in L Chain with RNOK203

The culture supernatant fractions of a wild-type RNOK203/its modifiedantibodies were diluted step-wise and compared for their expressionlevel of a functional Fab protein in ELISA. As a result, it was foundthat L15R modified RNOK203 antibody exhibited an increased expressionlevel (FIG. 15).

Example 28: Assessment of Tolerance Against Acid of Modified Antibodiesat Position 15 in L Chain with RNOK203

The culture supernatant fractions of wild-type RNOK203/its modifiedantibodies were diluted to 50 μL/tube with Block Ace and adjusted to pH5.0 with 1N HCl and a pH meter (HORIBA) and treated at 25° C. for 2hours. The samples were then adjusted to pH 7 with 1M Tris-HCl (pH 9.5)and subjected to ELISA where a ratio of the obtained absorbance to thatof untreated samples was shown as a residual activity in a graph. As aresult, it was found that L15R modified RNOK203 antibody exhibitedincreased tolerance against an acid (FIG. 16).

Example 29: Construction of Modified Antibodies at Position 15 in LChain of SEB3-2-7

It was investigated whether the same effects observed in Examples 20 to28 for the modification to Arg or Ser at position 15 in L chain couldalso be detected for SEB3-2-7 wherein the 15th amino acid residue isVal.

Using a wild-type Fab expression plasmid as a template, PCR wasperformed as described above for amplification of VL gene using OligoDNAs in which codon at position 15 in L chain was CGT(Arg) and TCT(Ser).The amplified VL gene was replaced for the VL region of a wild-type Fabexpression plasmid. JM83 was transformed with the resulting plasmid andthe DNA sequence was analyzed for the obtained clones to prove that theycontained the sequences as designed.

Example 30: Assessment of Heat Stability of Modified Antibodies atPosition 15 in L Chain with SEB3-2-7

The culture supernatant fractions of wild-type SEB3-2-7/its modifiedantibodies were diluted to 50 μL/tube with 1% BSA-PBS and treated in awater-bath at 42° C. for 2 hours. The samples were then restored at roomtemperature and subjected to ELISA where a ratio of the obtainedabsorbance to that of untreated samples was shown as a residual activityin a graph. As a result, it was found that V15R and V15S modifiedSEB3-2-7 antibodies exhibited increased heat stability (FIG. 17).

Example 31: Assessment of Tolerance Against Acid of Modified Antibodiesat Position 15 in L Chain with SEB3-2-7

The culture supernatant fractions of wild-type SEB3-2-7/its modifiedantibodies were diluted to 50 μL/tube with 1% BSA-PBS and adjusted to pH4.5 or pH 4.0 with HCl and pH meter (HORIBA) and treated at 25° C. for 2hours. The samples were then adjusted to pH 7 with 1M Tris-HCl (pH 9.5)and subjected to ELISA where a ratio of the obtained absorbance to thatof untreated samples was shown as a residual activity in a graph. As aresult, it was found that V15R and V15S modified SEB3-2-7 antibodiesexhibited increased tolerance against an acid (FIG. 18).

Example 32: Assessment of Heat Stability of Modified Antibodies atPosition 18 in L Chain with RNOK203

The culture supernatant fractions of wild-type RNOK203/its modifiedantibodies were diluted to 50 μL/tube with Block Ace and treated in awater-bath at 45° C. for 2 hours. The samples were then restored at roomtemperature and subjected to ELISA where a ratio of the obtainedabsorbance to that of untreated samples was shown as a residual activityin a graph for comparison between the modified antibodies. As a result,it was found that P18R, P18S and P18F modified RNOK203 antibodiesexhibited increased heat stability (FIG. 19).

Example 33: Assessment of Tolerance Against Acid of Modified Antibodiesat Position 18 in L Chain with RNOK203

The culture supernatant fractions of wild-type RNOK203/its modifiedantibodies were diluted to 50 μL/tube with Block Ace and adjusted to pH4.0 with 1N HCl and a pH meter (HORIBA) and treated at 25° C. for 2hours. The samples were then adjusted to pH 7 with 1M Tris-HCl (pH 9.5)and subjected to ELISA where a ratio of the obtained absorbance to thatof untreated samples was shown as a residual activity in a graph forcomparison between the modified antibodies. As a result, it was foundthat P18F, P18A, P18W, P18L, P18R, P18Q and P18S modified RNOK203antibodies exhibited increased tolerance against an acid. Typicalresults are shown in a graph (FIG. 20) whereas the results of theremaining modified antibodies are summarized in Table 2 for a ratio ofthe respective residual activity to that of the wild-type antibody.

TABLE 2 Comparison with wild- Modified antibodies type (%) P18F 180 P18A160 P18W 200 P18L 150 P18R 160 P18Q 170 P18S 140

Example 34: Assessment of Tolerance Against Freeze-Thawing of ModifiedAntibodies at Position 18 in L Chain with RNOK203

The culture supernatant fractions of wild-type RNOK203/its modifiedantibodies were subjected to freeze-thawing repeated for either sixtimes or only once and then to ELISA where a ratio of the absorbanceobtained from the samples subjected to six freeze-thawing procedures tothat of samples subjected to only one freeze-thawing procedure as aresidual activity. As a result, it was found that P18R, P18Q, P18S, P18Fand P18L modified RNOK203 antibodies exhibited increased toleranceagainst freeze-thawing (FIG. 21).

INDUSTRIAL APPLICABILITY

The method for improving the properties of a human antibody or ahumanized antibody according to the present invention is characterizedby that at least any one of the amino acid residues at position 8, 12,15 or 18 (according to Kabat numbering) in a light chain variable region(hereinafter referred to as “VL chain”) is substituted with a differentamino acid other than proline or cysteine. With substitution of merely asingle amino acid residue at such a specific position in an antibody, itis possible to obtain an antibody with an improved expression leveland/or stability. Accordingly, a human antibody or a humanized antibodywith an improved expression level and/or stability obtained by themethod according to the present invention, as possessing all thefeatures of high antigen specificity, low immunogenicity, highproductivity and improved stability, may be extremely useful in theclinical field such as human diagnosis and therapy as a specificantibody targeting a disease-related antigen.

The invention claimed is:
 1. A method for improving the expression leveland/or stability of a human antibody or a humanized antibody, saidmethod comprising: modifying a human antibody or a humanized antibody bysubstituting proline at position 12 (according to Kabat numbering) in alight chain variable region (hereinafter referred to as “VL chain”) ofthe human antibody or the humanized antibody with a different amino acidother than proline selected from any one of the amino acidsubstitutions: Ser, His, Val, Gly, Leu, Arg, Phe, Met or Glu, whereinsaid VL chain belongs to human Vκ2 family and has a framework FR1, whichprior to substitution, has the FR1 sequence encoded by SEQ ID:32 fromDPK18 (GeneBank Accession No. X63403), to thereby obtain a modifiedhuman antibody or a modified humanized antibody having improvedexpression level and/or stability as compared to a control, which isidentical to the human antibody or the humanized antibody but withoutsaid substitution.
 2. The method according to claim 1, wherein the FR1of the VL chain belonging to human Vx2 family after substitution has theamino acid sequence selected from the amino acid sequences as depictedin SEQ ID NO: 9 to
 17. 3. The method according to claim 1, wherein saidantibody is an intact antibody, or an antibody fragment such as Fab,Fab′, F(ab′)2, scAb, scFv, diabody [a recombinant dimer antibodyconsisting of homologous or heterologous heavy chain variable region (VHchain) and VL chain connected by a short linker peptide] or scFv-Fc; ora fused antibody or a fused antibody fragment with other proteins; or anantibody or an antibody fragment labeled with a low molecular weightcompound; or an antibody or an antibody fragment modified with a highmolecular weight compound.
 4. The method according to claim 1, whereinthe amino acid substitution is done by a genetic recombinationtechnique.
 5. A method for preparing an antibody with an improvedexpression level and/or stability according to claim 1, comprising:preparing a gene coding for an amino acid sequence of said humanantibody or said humanized antibody or a fragment of said human orhumanized antibody, each comprising the amino acid sequence of the VLchain after substitution; transforming a host of eukaryotic orprokaryotic organisms with said gene; expressing said human antibody orsaid humanized antibody or said human antibody fragment or saidhumanized antibody fragment from said host; and recovering said humanantibody or said humanized antibody or said human antibody fragment orsaid humanized antibody fragment.